Table 1_Resolution of MALDI-TOF compared to whole genome sequencing for identification of Bacillus species isolated from cleanrooms at NASA Johnson Space Center.docx

IntroductionBacteria are frequently isolated from surfaces in cleanrooms, where astromaterials are curated, at NASA’s Lyndon B. Johnson Space Center (JSC). Bacillus species are of particular interest because endospores can endure extreme conditions. Current monitoring programs at JSC rely on culturing microbes from swabs of surfaces followed by identification by 16S rRNA sequencing and the VITEK 2 Compact bacterial identification system. These methods have limited power to resolve Bacillus species. Whole genome sequencing (WGS) is the current standard for bacterial identification but is expensive and time-consuming. Matrix-assisted laser desorption - time of flight mass spectrometry (MALDI-TOF MS), provides a rapid, low-cost, method of identifying bacterial isolates and has a higher resolution than 16S rRNA sequencing, particularly for Bacillus species; however, few studies have compared this method to WGS for identification of Bacillus species isolated from cleanrooms.
MethodsTo address this, we selected 15 isolates for analysis with WGS and MALDI-TOF MS. Hybrid next-generation (Illumina) and 3rd-generation (nanopore) sequencing were used to draft genomes. Mass spectra, generated with MALDI-TOF MS, were processed with custom scripts to identify clusters of closely related isolates.
ResultsMALDI-TOF MS and WGS identified 13/15 and 9/14 at the species level, respectively, and clusters of species generated from MALDI-TOF MS showed good agreement, in terms of congruence of partitioning, with phylotypes generated with WGS. Pairs of strains that were > 94% similar to each other, in terms of average amino acid identity (AAI) predicted by WGS, consistently showed cosine similarities of mass spectra >0.8. The only discordance was for a pair of isolates that were classified as Paenibacillus species. This pair showed relatively high similarity (0.85) in terms of MALDI-TOF MS but only 85% similarity in terms of AAI. In addition, some strains isolated from cleanrooms at the JSC appeared closely related to strains isolated from spacecraft assembly cleanrooms.
DiscussionSince MALDI-TOF MS costs less than whole genome sequencing and offers a throughput of hundreds of isolates per hour, this approach appears to offer a cost-efficient option for identifying Bacillus species, and related microbes, isolated during routine monitoring of cleanrooms and similar built environments.

引言
美国国家航空航天局（NASA）林登·B·约翰逊航天中心（Lyndon B. Johnson Space Center, JSC）内，用于保藏天体物质的洁净室表面经常分离出细菌。芽孢杆菌属（Bacillus）物种尤为受关注，因其内生孢子可耐受极端环境。目前JSC的洁净室监测方案依赖于对表面拭子样本中的微生物进行培养，随后通过16S rRNA测序与VITEK 2 Compact细菌鉴定系统完成菌种鉴定。上述方法对芽孢杆菌属物种的区分能力有限。全基因组测序（Whole Genome Sequencing, WGS）是当前细菌鉴定的金标准，但该方法成本高昂且耗时较长。基质辅助激光解吸飞行时间质谱（Matrix-assisted laser desorption time-of-flight mass spectrometry, MALDI-TOF MS）是一种快速、低成本的细菌分离株鉴定方法，其分辨能力优于16S rRNA测序，在芽孢杆菌属物种鉴定中尤为突出；但目前鲜有研究将该方法与WGS用于洁净室分离芽孢杆菌属物种的鉴定对比。
方法
为解决上述问题，本研究选取15株细菌分离株，采用WGS与MALDI-TOF MS进行分析。采用二代测序（Illumina）与三代测序（纳米孔，Nanopore）技术进行混合组装，以获得细菌基因组草图。通过MALDI-TOF MS获取的质谱数据经自定义脚本处理后，可识别出亲缘关系相近的分离株聚类簇。
结果
MALDI-TOF MS与WGS分别在物种水平上鉴定出13/15与9/14株样本；基于MALDI-TOF MS构建的物种聚类簇，其聚类划分一致性与WGS生成的系统型（phylotypes）具有良好的一致性。经WGS预测，平均氨基酸同一性（Average Amino Acid Identity, AAI）高于94%的菌株对，其质谱余弦相似度均高于0.8。唯一不一致的结果来自一对被归类为类芽孢杆菌属（Paenibacillus）物种的分离株：该菌株对的MALDI-TOF MS质谱相似度为0.85，但其AAI仅为85%。此外，部分从JSC洁净室分离得到的菌株，与航天器装配洁净室分离得到的菌株亲缘关系相近。
讨论
相较于全基因组测序，MALDI-TOF MS成本更低，且每小时可完成数百株分离株的鉴定，因此该方法可为洁净室及同类人工建造环境常规监测中分离得到的芽孢杆菌属物种及相关微生物的鉴定提供一种高性价比的方案。